A Very Long-acting Exatecan and Its Synergism with DNA Damage Response Inhibitors

Exatecan (Exa) is a very potent inhibitor of topoisomerase I and anticancer agent. It has been intensively studied as a single agent, a large macromolecular conjugate and as the payload component of antigen-dependent antibody–drug conjugates. The current work describes an antigen-independent conjugate of Exa with polyethylene glycol (PEG) that slowly releases free Exa. Exa was conjugated to a 4-arm 40 kDa PEG through a β-eliminative cleavable linker. Pharmacokinetic studies in mice showed that the conjugate has an apparent circulating half-life of 12 hours, which reflects a composite of both the rate of renal elimination (half-life ∼18 hours) and release of Exa (half-life ∼40 hours). Remarkably, a single low dose of 10 μmol/kg PEG-Exa—only approximately 0.2 μmol/mouse—caused complete suppression of tumor growth of BRCA1-deficient MX-1 xenografts lasting over 40 days. A single low dose of 2.5 μmol/kg PEG-Exa administered with low but efficacious doses of the PARP inhibitor talazoparib showed strong synergy and caused significant tumor regression. Furthermore, the same low, single dose of PEG-Exa administered with the ATR inhibitor VX970 at doses of the DNA damage response inhibitor that do not affect tumor growth show high tumor regression, strong synergy, and synthetic lethality. Significance: A circulating conjugate that slowly releases Exa is described. It is efficacious after a single dose and synergistic with ATR and PARP inhibitors.


Introduction
DNA-damaging agents-long-standing mainstays of cancer chemotherapyhave taken on added importance as agents that enhance DNA damage response (DDR) defects and inhibitors in tumors. DNA damage in the face of impaired ability to repair damaged DNA can result in selective toxicity and synthetic lethality (1). An important target to cause-specific DNA damage is the enzyme topoisomerase I (TOP1), which is inhibited by camptothecin and its analogs (2,3). During DNA replication, TOP1 relaxes supercoiled DNA by forming a covalent phosphodiester with DNA, permitting the broken strand to rotate around and relax the TOP1-bound DNA, and then reversing covalent binding to religate DNA. In the presence of an effective TOP1 inhibitor (TOP1i) such as a camptothecin analog, the covalent TOP1i-DNA complexes are specifically trapped, forming a TOP1 cleavage complex (TOP1cc). Subsequently, single and double DNA strand breaks form that lead to cell death if unrepaired. Because 1 ProLynx, San Francisco, California.

SN-38
Exatecan (Exa) Another TOP1i of high current interest is the camptothecin analog exatecan (Exa), an even more potent TOP1 trapping agent/inhibitor than SN-38 (5,9); for a comprehensive review, see ref. 10. As a single agent, Exa progressed as far as phase III clinical trials before its development was discontinued because of dose-limiting side effects, and the absence of therapeutic benefits in combination with gemcitabine (11).
One of the limitations of current small-molecule TOP1i is their relatively short half-lives-the SN-38 formed from CPT-11 has an apparent t 1/2 of approximately 12 hours (8) and Exa has a t 1/2 of only approximately 10 hours in humans (12). As described above, effective suppression of TOP1 to cause DNA damage requires continuous presence of the inhibitor during S-phase of the cell cycle and inhibition of TOP1 is rapidly reversed upon drug withdrawal. Hence, the short half-lives of these TOP1i are suboptimal for drug efficacy, and extended-release forms of these agents are expected to show improved efficacy.
An approach toward obtaining longer-lived TOP1i that actively target specific tumors is to incorporate them as payloads in antibody-drug conjugates (ADC).
Indeed, three ADCs containing TOP1is-the Trop2-directed sacituzumab govitecan (Trodelvy) delivering SN-38, the HER2-targeted trastuzumab deruxtecan (Enhertu), and Trop2-directed datopotamab deruxtecan delivering a close derivative of Exa-show impressive results in the clinic and have been approved (13). However, most tumors do not have the Trop2 or HER2 surface antigens recognized by these ADCs, and are thus not suitable targets for these tumor-targeted agents. Likewise, ADCs cannot specifically target subsets of a tumor that differs from others in a TOP1i DDR defect, such as BRCA or ATM deficiency. Thus, a need remains to develop long-acting, tumor-agnostic TOP1i prodrugs that do not require the presence of specific tumor antigens to express their antitumor effects.
Another approach toward obtaining long-lived TOP1i has been to develop passively targeted antigen-independent prodrugs in which the TOP1i is attached to a tumor-accumulating macromolecular carrier by a releasable linker. Depending on the linker used the TOP1i may be slowly released from the prodrug systemically and/or after penetrating the tumor by the EPR effect. For example, DE-310 is a conjugate of a large 340 kDa CM-dextran polyalcohol carrier covalently linked to Exa by a cathepsin-sensitive peptidyl spacer (14). The conjugate has a long systemic t 1/2 (15,16) and passively accumulates in xenografts where, after cell internalization, lysosomal cathepsins cleave the linker to release Exa (17). Although DE-310 suppressed growth of xenografts for long periods (14), accumulation of the prodrug in human tumors was not observed (18) and sub-sequent efforts with Exa were apparently redirected toward its use as a payload in ADCs. It would be advantageous to have a TOP1i prodrug that can passively target tumors but does not mandate internalization and proteolysis for activity.
We have had an interest in developing a general approach for half-life extension of therapeutics in which a drug is covalently tethered to a long-lived carrier by a linker that slowly cleaves by β-elimination to release the drug (Scheme 1; ref. 19). The cleavage rate, k 1 , is determined by the nature of an electron-withdrawing "modulator" (Mod) that controls the acidity of the adjacent C-H bond, and is unaffected by enzymes or general acid/base catalysts. A carrier used for βeliminative linkers is often a long-lived circulating macromolecule-such as high molecular weight polyethylene glycol (PEG). For this purpose, 4-arm PEG 40kDa -a 15-nm-diameter nanocarrier-may be optimal because smaller PEGs have shorter half-lives, while larger PEGs all show similar elimination rates. The prodrug is usually eliminated with a t 1/2 similar to that for carrier, and the apparent t 1/2 of the released drug is usually similar to the t 1/2 of the prodrug. Furthermore, tumor accumulation occurs because of the long circulating t 1/2 and near-ideal size and shape of the 15-nm-diameter nanocarriers to penetrate large pores of tumor vasculature (20,21).
We used this technology to prepare the passively-targeted PEG 40kDa -SN-38 conjugates PLX038 and PLX038A that spontaneously slowly release the potent TOP1i SN-38 by a β-elimination reaction (22,23). The favorable properties of these prodrugs are (i) a long t 1/2 of released SN-38 approximating the t 1/2 of the PEG 40kDa carrier with concomitant benefits of low C max and prolonged exposure, (ii) high accumulation and retention in tumors (20,22), and (iii) a high therapeutic effect in tumors with DDR defects and in combination with DDR inhibitors (22). Hence, PLX038 provides a prolonged duration of DNA damage to achieve synthetic lethality of DNA repair deficient or inhibited tumors.
The objective of the current work was to design an efficacious long-acting PEG-Exa prodrug analogous to PLX038 that slowly releases Exa but-unlike ADCs with a TOP1i payload-is not limited to cancers expressing high levels of a specific tumor antigen. Here, we (i) describe the synthesis and characterization of a PEG-Exa prodrug, (ii) ascertain its pharmacokinetics in mice, and (iii) determine antitumor effects of the conjugate in mouse xenografts. We report that a single injection of the PEG 40kDa -Exa provides effective and long-lasting antitumor effects in a BRCA1-deficient mouse xenograft and shows synergy with PARPi talazoparib (TLZ) and the ATR inhibitor (ATRi) VX970.

. Stable PEG-Exa B
Stable PEG-Exa was prepared in a similar fashion as above except 6-(Bocamino)hexyl N-(hydroxysuccinimidyl) carbonate was used as the linker.  Individual C versus t curves for each mouse were fit to a single exponential decay with 1/C 2 weighting using data obtained from 24 to 120 hours. t 1/2 values obtained for each mouse were combined to obtain a mean and SD for each group of animals. Tumor growth (TG) and tumor growth inhibition (TGI) were calculated as described previously (25) Table S3 shows the values for synergy for 3A and the ATRi VX970.

Data Availability
Data were generated by the authors and available on request.

Synthesis and In Vitro Characterization of 4-arm PEG 40kDa -Exa Conjugates
The releasable PEG 40kDa -Exa conjugate 3A and its stable counterpart 3B were prepared by methods used for analogous carbamate conjugates (Scheme 2;   Figure 2 shows the C versus t plot after intraperitoneal injection of releasable PEG 40kDa -Exa 3A and stable PEG 40kDa -Exa 3B in the mouse; Table 1 shows the

Tumor Inhibition by PEG-Exa
It has been reported that a total dose of 25 and 75 mg (57 and 170 μmol)/kg Exa administered as administered four times at 4-day intervals caused regres-sion of BRCA1-deficient MX-1 TNBC xenografts at 28 days (26). Figure 3 shows the effect of varying single intraperitoneal doses of 2.5 to 15 μmol/kg PEG-Exa 3A on inhibition of growth of the MX-1 TNBC xenografts. As shown, single doses of 10 and 15 μmol/kg PEG-Exa caused complete growth inhibition of tumors without weight loss of hosts for >48 days (Fig. 3). Note that the single 10 μmol/kg PEG-Exa dose is 6-and 18-fold lower than the dose of free Exa required to suppress MX-1 xenografts (26). The growth inhibition for 2.5 μmol/kg PEG-Exa is nearly identical to that of 15 μmol/kg PEG-SN-38 (PLX038A) indicating that PEG-Exa is approximately 6-fold more potent than PLX038A in growth suppression of this tumor; the in vitro antiproliferative activity of Exa has also been reported to be significantly more potent than SN-38 (5,27).

Tumor Inhibition by PEG-Exa Combinations with TLZ or VX970
We measured the effects of a combination of PEG-Exa with the PARPi TLZ and the ATRi VX970 on growth of MX-1 xenografts. TG and TGI were calculated as described previously (25) Table S3). Hence, doses of VX970 that do not affect tumor growth strongly act synergistically to enhance the chemotherapeutic activity of PEG-Exa.

Discussion
We have been developing antigen-independent, long-acting prodrugs of TOP1i with the objective of optimizing DNA-damaging agents for use with DDR deficiencies or with DDR inhibitors. Such agents are differentiated from an ADC TOP1i in that they target tumors with DDR defects rather than antigen-specific tumor types. Synthetic lethality can be achieved when TOP1i are used in DDRdeficient tumors and/or in combination with an appropriate DDRi; the TOP1i creates DNA damage and the DDR defect or DDRi inhibits repair. Because synergy of the TOP1i and a DDRi requires concurrent presence of DNA damage and the DDRi, we posit that a prolonged duration of DNA damage would be permissive of more frequent and/or more intense DDRi dosing than possible with currently available short-acting TOP1i.
We previously reported studies on PLX038, a long-acting prodrug of SN-38, composed of a 4-arm 40 kDa PEG attached to SN-38 by releasable linkers that slowly cleave to release the drug. The prodrug and released SN-38 have long t 1/2 values of 5 days in humans, approximately 10-fold longer than the SN-38 released from CPT-11. Also, the small 15 nm nanomolecules readily penetrate large pores of tumor vasculature, and accumulate and are retained in the tumor microenvironment for long periods (20). PLX038 is currently being studied in clinical trials (NCT05465941; NCT04209595).
The current study focuses on Exa, another potent inhibitor of TOP1 with potential benefits over CPT-11/SN-38 (5). First, it is even more potent than SN-38 as a TOP1i, which may result in less-off target effects. Second, because it is not extensively metabolized, there should be less interpatient variability than with CPT-11. Finally, Exa does not appear to be a substrate for drug-efflux pumps that may cause resistance to CPT-11/SN-38 (27,30) The objective of this work was to develop a long-acting prodrug of Exa that benefits from improved pharmacokinetics and pharmacology. The specific aims were to (i) develop chemistry that enables attachment of Exa to long-lived carriers via β-eliminative linkers, (ii) determine the pharmacokinetics of PEG-Exa in mice, and (iii) ascertain the antitumor activity of the releasable PEG-Exa 3B in mouse xenografts.
To prepare the PEG-Exa conjugates, we coupled Exa with appropriate linker carbonates to provide both stable and β-eliminative releasable amino-linker- We pondered why the antitumor effects of a single low dose of PEG 40kDa -Exa 3A in mouse xenografts are so long lasting. First, the high exposure of released Exa over prolonged periods should have profound antitumor effectsmuch greater than intermittent dosing of free Exa. Second, tumor cells may become significantly more sensitive to a drug as a function of the time of exposure (31,32); indeed, the inhibitory potency of Exa increases about 80-fold upon prolonged exposure (30). Here, the lower Exa concentration requirements of an increasingly-sensitive tumor over time would counter the reduced concentration that occurs over time by renal elimination. Finally, large PEGylated prodrugs can accumulate and be retained in tumors, releasing the cargo in the tumor environment over long periods. Indeed, the analogous PEG prodrug of SN-38, PLX038A (20), has a high accumulation of approximately 10% of the initial dose in the MX-1 xenograft and very long efflux t 1/2 of 17 days.
It has been well established that a combination of a TOP1i and an appropriate DDRi partner can be highly synergistic in many DDR-deficient tumors. An obvious DDRi partner for a long-acting TOP1i would a PARPi, and the BRCA1deficient MX-1 tumor used here is highly sensitive to both drugs. Indeed, as with PLX038 (28), PEG-Exa is synergistic with the PARPi TLZ. However, despite promising preclinical data such as reported here, TOP1i and PARPi combinations have proven challenging in the clinic where myelosuppression has required large dose reductions of both single agents (3). It has been proposed that modification of the interval between dosing a long-acting, nanomolecular TOP1i and a PARPi might avoid overlapping toxicities (3) and this hypothesis warrants testing in the clinic.
Another candidate DDRi partner for a long-acting TOP1i is an ATRi. Pommier and colleagues identified ATR as a synthetically lethal gene for TOP1is (33), and showed that at nontoxic doses ATRi are strongly synergistic with conventional short-acting TOP1i-including Exa (5,33). Furthermore, in a phase II trial of relapsed small cell lung cancer, combination of the TOP1i topotecan with the ATRi VX-970 showed a high 36% ORR and acceptable side effects even though administration of both drugs was confined to a 5-day interval of a 21-day cycle (34). We speculate that the prolonged DNA damage expected from a long acting TOP1i in a DDR-deficient tumor would allow more frequent dosing of an ATRi to push tumors beyond their survival threshold.
On the basis of these reports and rationale, we tested the effect of the ATRi VX970 in combination with long-acting PEG-Exa in BRCA-deficient MX-1 xenografts. First, we showed that a relatively high dose of 64 μmol/kg of VX970 for 4 consecutive days a week had no effect on growth of MX-1 xenografts in spite of their BRCA deficiency. Then, we showed that a combination of the same ineffectual dose of VX970 with a single administration of a low 2.5 μmol/kg of PEG-Exa resulted in strong synergy. Hence, the combination of a single low-dose of a long-acting TOP1i with low doses of ATRi are synergistic and synthetically lethal, and should enable a flexible dosing schedule with maximum overlapping exposure of both drugs.
In summary, we developed a long-acting PEGylated prodrug of Exa. A single injection of the prodrug is highly effective in suppressing the BRCA1-deficient MX-1 xenograft for prolonged periods. In addition, PEG-Exa shows synergy with the PARPi TLZ and the ATRi VX970, which provide effective combinations worthy of further studies.